Active/passive immunization of the internal female reproductive organs

ABSTRACT

Antibody or antigen containing microparticles for the active or passive immunization of the internal female reproductive organs, comprising: microparticles of an antigen or antibody incorporated in a matrix material which is biocompatible and biologically degradable, said microparticles capable of being transported after deposition in the vagina by the natural transport mechanism of the internal female reproductive organs across the cervix into the uterus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 121,480,filed Feb. 14, 1980 which in turn is a continuation-in-part ofapplication Ser. No. 952,109, filed Oct. 17, 1978, now both abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for introducing therapeutic ormedicinal agents into the uterus and fallopian tubes. More particularly,the invention relates to a method of eliciting an active or passiveimmunization response in the internal female reproduction organs byintroducing microparticles containing a specific antibody or antigeninto the vagina and allowing the microparticles to be drawn through thecervix into the uterus.

2. Description of the Prior Art

In the past, the methods of generally treating the internal reproductiveorgans of the female have included principally the oral ingestion or theinjection of drugs into the patient in order to treat diseases and toregulatd the female reproductive cycle. No methods are known of treatingthe uterus and fallopian tubes by introducing a drug directly into thevagina where the natural transport mechanism of the internalreproductive organs conveys the drug across the cervix into the uterus.Yet, such a direct technique of locally introducing drugs into thevagina would be highly advantageous from the viewpoint of rapidly andeffectively conveying drugs across the cervix into the uterus. Thistechnique would be especially useful for the delivery of biologicallyactive substances, such as antibodies and antigens, directly into theuterus to increase the level of immunization of the femal reproductiveorgans. Because systemic antibodies are not secreted by the internalreproductive organs, the immunization levels of these organs cannot beincreased by systemic administration of either antibodies or antigens,but rather must be increased by local administration of antibodies orantigens to the female reproductive organs.

In the past various drugs and cosmetic agents have been encapsulated inthe form of microcapsules for the purpose of delivering these agents tothe vagina by slow, sustained release of the agent from themicrocapsules. However, these techniques have only been useful in thetreatment of the vagina and not the other internal female reproductiveorgans. For example, Zaffaroni in U.S. Pat. No. 3,921,636 shows a drugdelivery device in which microcapsules containing a medicinal agent areincorporated in a carrier device such as a tampon, sanitary napkin orintrauterine device. Thus, a tampon containing microencapsulatedcontraceptive hormone can be inserted into the vagina and the hormonewill be gradually released by dissolution of the microcapsules. Sincethe hormone is released in the vagina, the vagina is the site in whichthe hormone is absorbed by the body. This technique does not provide ameans of delivering drugs to the uterus by transport across the cervix.

U.S. Pat. No. 3,918,452 shows a technique in which a contraceptive agentis delivered to the vagina by inserting a tampon containingmicrocapsules composed of a contraceptive composition into the vagina.The contraceptive agent such as a spermicide is then released slowlywith time into the vagina where the contraceptive agent has its effect.In this technique the effects of the contraceptive agent are limitedonly to the vagina and not to any other portions of the internal femalereproductive organs.

Because it would be highly desirable to be able to introduce medicinalagents or therapeutic agents directly into the uterus and Fallopiantubes by transport of said agents across the cervix, various techniqueshave been attempt to achieve this end. One approach that has beensuggested is to encapsulate a medicinal or therapeutic agent in the formof microcapsules and then deposit the microcapsules in the vaginawhereupon the microcapsules are transported. As a result of some earlyinvestigations, it is known that carbon particles from a cap containinga suspension of carbon particles, when placed over the cervix, can berecovered from the uterus after coitus, as shown by Amersbach,"Sterilitat Und Frigiditat," Munchen. Med. Wchnschr. 77: 225, 1930. Thisshows that nonmotile particles migrate in the female reproductive tract.It was also demonstrated by J. Trapl, "Neuve Anschauunger uber den Ei-und Samentransport in den Geschlechtsteilen de Frau," Zentralbl. Gynak.67: 547, 1943, that even without the use of a cervical cap, carmineparticles migrate thus demonstrating that nonmotile particles other thancarbon also migrate.

Still other investigators, R. Krehbiel and H. P. Carstens, "RoentgenRabbit", Am. J. Physiol. 125: 571, 1959, have shown that the passage ofthe radio-opaque oil, when placed in the vagina of a rabbit was blockeduntil after the vulva was stimulated. Stimulation of the vulva causedcontraction waves which transported the oil into the uterus and up intothe uterolubal junction within several seconds. Other earlierinvestigations found that graphite and dyes in gelatin were nottransported whether applied to the vagina before or after copulation,but carmine particles in cocoa butter were transported to the uterus andtubes. The implication of the data is that the nature of the particlesaffects the transport process and that transport is assisted by muscularcontractions. Hartman, in "How Do Sperms Get Into the Uterus?" Fertil.and Steril 8: 403, 1957, concluded that the transport of sperm in thereproductive tract, transport occurs principally by cooperation of theparticles with the musculature of the female reproductive tract. He alsoconcluded that the function of the flagellum of the sperm is to aid inthe penetration of the head of the sperm into corona radiata, the zonapellucida and the vitelline membrane of the ovum. G. M. Duncan and D. R.Kalkwarf, "Sustained Release Systems for Fertility Control," in HumanReproduction: Conception and Contraception, edited by E. S. E. Hafez andT. N. Evans, Harper and Row, New York, 1973, have concluded fromexperiments that non-motile particles which are about the size of thehead of the sperm migrate directionally through the cervix to thefallopian tubes. Thus, the reference indicates that non-motile particlesof a size of 5 μm or less migrate throughout the internal reproductiveorgans when introduced into the vagina. However, when microcapsules ofprogesterone encapsulated within a suitable wall material such ascellulose acetate butyrate and of a size ranging from 5 to 1400 μm wereintroduced into the vagina, the microcapsules did not migrate across thecervix into the uterus, but rather were transported in the reversedirection. Therefore, the reference clearly suggests that microcapsulesof a size greater than 5 μm will not migrate inward to the internalfemale reproductive organs.

A need therefore, continues to exist for a method by which variousdisorders and diseases of the internal female reproductive organs can belocally treated by applying microparticles of various medicinal andtherapeutic agents to the vagina and allowing the natural transportmechanism of the organs to draw the microparticles across the cervixinto the uterus where the medicinal or therapeutic agent is delivered tothe uterus and other internal organs.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodby which medicinal and therapeutic agents can be locally administered tothe vagina and transported through the cervix into the uterus to treatthe internal female reproductive organs.

Another object of the present invention is to provide a method by whichantigens or antibodies can be delivered to the uterus so that theinternal organs can be treated directly to avoid systemic administrationof the antigen or antibody.

Still another object of the present invention is to provide antibodiesand antigens incorporated within microparticles which, when deposited inthe vagina, can be transported across the the cervix into the uterus bythe natrual transport mechanism of the internal reproductive organs.

Briefly, these objects and other objects of the present invention ashereinafter will become more readily apparent can be attained by amethod for achieving the passive immunization of the internal femalereproductive organs by depositing antibody containing microparticlesdirectly into the vagina and allowing the natural transport mechanism ofthe internal organs to convey the microparticles across the cervix intothe uterus, whereby the antibody is continuously released from themicroparticles.

The present invention can also be used to effect the active immunizationof the internal organs by using microparticles which contain an antigen.The microparticles employed in the present process contain the antigenor antibody in a matrix which is biocompatible and biologicallydegradable.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows microparticles of a monolithic structure containing apharmaceutical agent;

FIG. 2 shows microparticles formed of a core of pharmaceutical agent ina matrix material surrounded by a shell of matrix material;

FIG. 3 shows microparticles formed of a core of pharmaceutical agentsurrounded by a shell of matrix material;

FIG. 4 shows microparticles of an onion-skin structure of alternatinglayers of matrix material and pharmaceutical agent; and

FIG. 5 shows microparticles formed of a core of one particularpharmaceutical agent surrounded by a shell of matrix material containinga second type of pharmaceutical agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The principal object of the present invention is to provide a method fordelivering various antibodies or antigens directly to the internalreproductive organs to obviate systemic introduction of antigens orantibodies for the treatment of the reproductive organs. Systemicintroduction, in fact, cannot be used as a means for administering manyantigens and antibodies into the body for treatment of the reproductiveorgans. An antibody is defined as any body of globulins that combinespecifically with antigens and neutralize toxins, agglutinate bacteriaor cells, and precipitate soluble antigens. Antibodies are produced bythe specialized cells of the endothelial reticular immune system inresponse to a challenge by antigens. The antibodies elicited by thechallenge are highly specific to the antigen which evokes the repsonse.This characteristic of antibodies has led to their exploitation inclinical medicine for both the diagnosis and treatment of disease.Moreover, both active and passive immunization have been used as acommon therapeutic approach for the prevention and treatment ofinfections diseases in man and animals.

In the past specific antibodies have been employed to retard the growthand proliferation of pathogenic microorganisms in both man and animalsas well as to neutralize various bacterial toxins. Antibodies have alsobeen used to control the growth and development of tumors and tomodulate the immune system. Other medical uses of antibodies include theneutralization of the biological activity of drugs and hormones andlocalization of cells that convey unique antigens which may beassociated with pathogenic conditions.

There are two basic ways in which the role of antibodies can bestimulated in the body to counteract the effects of antigens. Onetechnique is active immunization while the other is passiveimmunization. In order to actively immunize a subject, the subject isadministered an antigen to induce the formation of endogeneousantibodies. Normally, this technique requires up to two weeks before asufficiently good level of antibody response is achieved. Because of thedelay involved, the active immunization technique imposes limitationsfor the treatment of infectious diseases which have a short incubationtime, for the treatment of a disease actively in progess and forreversing or modifying the effects of drugs, toxins, hormones, andenzymes. Furthermore, if active immunization is to be effective, thesubject must have at least a functioning immune system which is capableof repsonding to the invading antigen. Thus, patients suffering from animmunodeficiency disease are precluded from active immunization. Yet afurther restriction on the use of active immunization is that theantigens used to immunize a subject must be safe and non-toxic. The use,therefore, of toxic substances in the preparation of vaccines intendedfor human use is precluded.

The second basic immunization technique is passive immunization wherebyantibodies are administered in order to achieve temporary immuneprotection. Passive immunization has the advantage that the biologicaleffects are immediate and can be effectively used in patients sufferingfrom immunodeficiency diseases. Moreover, active immunization is notlimited to the use of non-toxic antigens because animal species can beused as the source for the protective antibodies.

The present invention provides a method of eliciting an active orpassive immunization response in the internal reproductive organs of afemale subject against an invading antigen as a result of the directintroduction of an antibody or antigen. The direct administration ofthese biologically active substances is achieved by incorporatingspecific antibody or antigen in microparticles and then introducing themicroparticles of antibody or antigen into the vagina of a femalesubject and allowing the natural transport mechanism of the internalorgans involving in particular the cervix and uterus to convey themicroparticles across the cervix into the uterus and eventually into thefallopian tubes. The microparticles release the antibody or antigen overa given period of time thus ensuring the presence of antibody or antigenin the reproduction organs which effectively provides for the active orpassive immunization of the subject administered the microparticles.

In the technique of the present invention microparticles containingantigen or antibodies are deposited in the vagina. Transport of themicroparticles is controlled by the cyclic changes in the endogeneousovarian steroid hormones, estradiol and progesterone. During the first14 days of the menstrual cycle or the follicular or estrogenic phase ofthe cycle, the ovaries produce estradiol which has a stimulating effecton the cervical muscle contraction. The frequency and amplitude of thecervical contractions during the follicular phase steadily increase fromday zero to day 14 at which time ovulation occurs and the cycle entersthe lutial phase or progestational phase when the ovaries begin tosecrete progesterone. Progesterone has an inhibitory effect on thecontractile activity of the cervix or alternatively a muscle relaxingeffect on the cervix. The ovarian hormones also exhibit an opposingeffect in the cells of the cervix in that estradiol causes anaccumulation of secretory products in the cells of the cervix whileprogesterone promotes the release of these products in the cervicallumen. The interactions described are the mechanism by which changes inthe viscosity of cervical fluid occur.

The fluid or mucous of the cervix is dynamic and is an aqueous type ofhydrogel. The transport of microparticles as well as sperm is dependentupon the permeability of the cervical mucous to the microparticles aswell as the propulsion provided by estrogen induced contractions of thecervix. The most appropriate time for transport of microparticles acrossthe cervix occurs when the uterus exhibits maximum contractile activityand cervical mucous permeability. Accordingly, the greatest rate oftransport of sperm or microparticles through the cervix should occurbetween day 12 and day 16 of the menstrual cycle, although the actualday of ovulation may vary from four to six days in different individualswith some transport occurring in some persons before day 12 of thecycle. Normally, the cervix is not very receptive to transport duringthe first twelve days of the menstrual cycle as well as between days 16and 28. Thus, in order to deliver microparticles containing antigen orantibody into the cervix, the microparticles in an appropriate dosageneed only to be deposited in the vagina prior to day 16 of the cycle,preferably before day 12 so that full advantage can be taken of the 4day period at midcycle for transport of the microparticles across thecervix into the uterus.

In a major embodiment of the present invention advantage can be taken ofthe fact that estrogen and progestin hormones have a transportstimulating effect on the internal organs. Thus, microparticlescontaining either an estrogen or progestin or both can be introducedinto the vagina to regulate the menstrual cycle of a woman who is notcycling regularly or to stimulate the cervix to transport microparticlesacross the cervix into the uterus and fallopian tubes. Once themicroparticles containing cycle regulating hormone are deposited in thevagina, hormone is released by the microparticles and is absorbed by thevagina and/or in the cervix if indeed some microparticles havepenetrated into the cervix. The locally absorbed hormone then inducesthe necessary secretory changes in the endometrium of the cervix andpromotes the contractile activity of the cervix necessary formicroparticles transport. Once transport activity has begun, themicroparticles are conveyed across the cervix into the uterus. From theabove discussion it is evident that microparticles containing cycleregulating hormone can be administered to regulate the menstrual cycleand to stimulate transport microparticles containing antigen or antibodyacross the cervix into the uterus.

The amount of estrogen or pregestin administered via the microparticlesshould be an amount which, when delivered locally by absorption, issufficient to regulate the menstrual cycle and to cause the change inthe cervix which results in microparticle transport across the cervix.In other words, as the menstrual cycle regulating hormone is absorbed bythe tissues of the vagina and indeed the tissues of the cervix if anymicroparticles have entered into the cervix, the desired regulatoryeffect is initiated immediately. The desired changes occur in the cervixwhich promote microparticles transport among which is a gradual increasein the contractile activity of the cervix. The administration of a largedose which results in achieving significant levels of the hormone on asystemic level, i.e. significant concentrations on the blood streamwhich result in delivery of the hormone to other organs such as thebrain and liver in addition to the reproductive organs, should beavoided. Both estrogen and progestin in proper amounts will stimulatetransport. However, a progestin at too high a level of concentrationwill have an adverse effect on microparticle transport because theprogestins have a muscle relaxing effect on the tissues of the cervix.However, the adverse effect of the administered progestin can bereversed by the administration of a sufficient concentration of anestrogen via estrogen containing microparticles such as estradiol whichas discussed supra induces contratile activity of the tissues of thecervix. Normally, when estradiol is delivered locally by way of themicroparticles, the amount of microparticles administered should besufficient to deliver from 0.1 to 1 mg per day over a 7-14 day period.On the other hand, when progesterone is incorporated in themicroparticles, the amount of microparticles administered should besufficient to deliver from 0.5 to 2 mg per day over a 7-14 day period.

As alluded to above the microparticles of the present invention canprovide the feasibility of locally administering a contraceptive agentto the cervix while simultaneously exogenously activating a menstrualcycle in a non-cycling woman by the administration of appropriateovarian hormone and stimulating the cervix for microparticle transport.Hence, estrogen containing microparticles which also contain the desiredantibody or antigen agent can be administered such that estradiol or asynthetic estrogen is released at the cervix for a fourteen day periodthus duplicating the first half of the menstrual cycle. When transportof the microparticles occurs across the cervix, antigen or antibody inthe microparticles is delivered to the uterus. Fourteen days afteradministration of the estrogen containing microparticles, progesteronecontaining micrparticles optionally containing antibody or antigen arethen administered. Thus, the complete natural menstrual cycle can beduplicated while providing antibody or antigen protection. It is alsoapparent that antigen or antibody and estrogen or progestin rather thanbeing incorporated in the same microparticles can be incorporated inseparate microparticles and delivered as a mixture so that eachbiologically active agent is present to deliver its intended function.Of course, it is also within the scope of this invention to delivermicroparticles containing estrogen or progestin into the cervix toregulate the cycle and thereafter administer antigen or antibodycontaining microparticles at the period of the cycle when the cervix isreceptive to transport. In the artificially induced cycle maximumtransport across the cervix is achieved between days 12 and 16. Sincethe cycle regulatory hormones are administerd locally in the presenttechnique, effective estradiol activity can be achieved at dosage ratesbetween 0.01 and 0.07 mg per day, while effective progesterone activitycan be attained at dosage rates of 0.04 to 0.14 mg per day. Estradioland progesterone are the regulatory hormones of choice because they arenaturally occurring endogenous hormones and therefore present notoxicity problems. However, it is evident that other well knownsynthetic estrogens and progestins can be employed as substitutes forestradiol and progesterone, respectively. Suitable estrogens includeestrone, mestranol, ethinyl estradiol, 2-methoxyestrone,2-hydroxyestrone and estriol. Suitable progestins include norethindrone,dimethisterone, ethynodiol diacetate, norethynodiol, norethindroneacetate and norgestrone. When the synthetic compounds are employed, thedose employed depends entirely upon the biological potency of thesynthetic estrogen or progestin compound.

Microparticles containing antibody or antigen and/or menstrual cycleregulating hormone can be formed in a variety of configurationsdepending upon such factors as when during menstrual cycle themicroparticles are delivered, whether sustained slow release or fastrelease of drug is desired, whether antigen or antibody is to beadministered simultaneously with the menstrual cycle regulating hormoneor after administration of the cycle regulating hormone, whether drugrelease is desired slowly and continually, intermittently or suddenly orthe like. In perhaps the simplest situation as shown in FIG. 1microparticles of a monolithic structure are prepared in which thedesired antigen or antibody 2 is distributed throughout a matrixmaterial 1 which is biodegradable and biocompatible. Once themicroparticles are deposited in the vagina they begin to slowlydeteriorate thereby continuously releasing the desired drug to achievethe desired daily dosage of drug over a prolonged period of time fromthe time they are deposited in the vagina until well after themicroparticles have been conveyed across the cervix and deposited in theuterus. In fact, the transport forces will result in some microparticlesbeing deposited in the fallopian tubes. The term microparticles is usedin a generic sense since the particles no matter in what particularconfiguration or regimen administered do not have to be sphericallyshaped in the form of microcapsules but can be of an irregular shape.Successful transport of the particles across the cervix is not dependenton the shape of the particles. When microparticles of a monolithicstructure are administered, the drug diffuses out of the microparticlesby gradual deterioration of the matrix material, by permeation of thedrug out of the matrix, or by both mechanisms.

In another embodiment of the microparticle structure, as shown in FIG.2, microparticles of a monolithic structure as shown in FIG. 1 areformed as described above. The monolithic microparticles are thenfurther processed such that a wall or outer shell 3 of matrix materialfree of drug is formed on each microparticle. This type of microparticleconfiguration is desirable where release of the drug is to be delayedfor some period of time after deposition of the microparticles in thevagina. The delayed release of drug obtained by using the abovemicroparticles, for instance, would allow sufficient time for themicroparticles to be deposited in the vagina, transported across thecervix and deposited in the urterus before the microparticlesdeteriorate to the point where the outer shell is essentially eliminatedand drug release commences. While the thickness of the outer shell canbe varied to any thickness desired, nevertheless, the overall size ofthe microparticles must be such that the microparticles possess spermsurrogate activity. If the microparticles are of such a relatively largesize that they do not have sperm surrogate activity, then themicroparticles will not be transported across the cervix and thereforecannot be used.

In still another embodiment of the microparticles as shown in FIG. 3,the microparticles can be designed for the sudden release of a largeamount of antibody or antigen. To achieve this purpose themicroparticles can be formed such that a core 5 of antigen or antibodyis encapsulated in a shell matrix material 1. Microparticles containinga core of drug would be particularly well suited in situations where anendogenous factor for disrupting the outer shell of the microparticlesis exploited. For example, the difference in pH of the mucosal fluids inthe vagina on the one hand, and the cervix and uterus on the other hand,can be exploited such that deterioration of the outer shell occurs whenthe microparticles reach the area of the cervix or uterus. In thissituation, the acidic pH of the vagina would have little or no effect onthe shell of the microparticles. However, when the microparticles areconveyed into the cervix where they are exposed to the neutral pHtherein, breakdown of the outer shell would commence eventuallyresulting in the sudden release of drug. This procedure would beparticularly desirable where it is desired to administer a boosterresponse after an individual has already received a primaryimmunization. Although it is more desirable to have a sustained releaseof antigen from microparticles of a monolithic structure, for instance,when an individual is to experience primary immunization, a shorterperiod of drug delivery is satisfactory for boosting the primary immuneresponse.

With regard to passive immunization by the administration of antibody,it would be very desirable when a subject or patient has an acuteinfection or high concentration of toxin to be able to deliver asubstantial amount of antibody quickly to the affected organ(s). Byadministering microparticles containing cores of antibody, once themicroparticles are conveyed into and through the cervix, the antibodywill suddenly be released in large quantities to counter the particulardisorder. After the initial treatment microparticles could beadministered to provide a sustained, lower level release of antibody tocontinue treatment.

In the treatment of patients for some disorders it is advantageous to beable to administer antigen or antibody in an intermittent fashion. Thiscould be accomplished by the use of microparticles having theconfiguration shown in FIG. 4 where alternate layers of drug alone ordispersed in matrix material 7 and drug free matrix material 1 areformed in concentric layers. When such microparticles are deposited inthe vagina, release of drug does not occur until the outer layer of themicroparticles disintegrates. This could delay drug release until themicroparticles are conveyed into the cervix and/or uterus. Once theunderlying layer is exposed drug release starts and continues until thelayer disintegrates or releases the drug. Drug release ceases as thenext underlying drug free layer is reached. In this manner intermittentrelease of the drug is achieved. An example of the applicability of thistechnique can be found in active immunization where the outermost druglayer releases antigen for a sustained period which is followed by aperiod for instance of a week or two, in which no drug is released.After the non-drug containing layer disintegrates, a second period ofantigen release starts. In this manner one could in a singleadministration of microparticles provide a primary immunization dosefollowed by a booster dose.

When it is desired to not only convey an antigen or antibody to theuterus through the cervix but also administer cycle regulatory hormonein order to activate or regulate the natural transport mechanism, it ispossible to administer microparticles of a monolithic structure as shownin FIG. 1 in which both antigen or antibody and cycle regulating hormoneare dispersed through a matrix material. In this manner, once themicroparticles are deposited in the vagina, release of both hormone andantigen or antibody starts and eventually the microparticles areconveyed across the cervix into the uterus. A perhaps more selectiveregimen of administration could be provided by microparticles which havean outermost matrix layer containing cycle regulatory hormone and aninner core of matrix material containing antigen or antibody. When suchmicroparticles are administered, the sustained release of cycleregulatory hormone occurs, and when the cervix is receptive totransport, the microparticles are transported across the cervix into theuterus. Release of the antigen or antibody will occur in the cervix oruterus as the underlying antigen or antibody core of the microparticlesis exposed. FIG. 5 shows microparticles of the structure discussed abovein which outer cycle regulatory hormone containing layer 9 encapsulatesinner antigen or antibody containing core 11. The antigen or antibodyalone can constitute the core of the microparticles or the antibody orantigen can be dispersed in matrix material to form core 11. However,outer layer or shell 9 is formulated by dispersing a menstrual cycleregulatory hormone in a matrix material.

From the above discussion it is evident that antibody or antigen aloneor in combination with a cycle regulatory hormone can be incorporated inmicroparticles in a variety of configurations depending upon how thedrug or drugs are to be released. Moreover, while multi-layeredmicroparticles such as the types shown in FIGS. 2, 4 and 5 are normallyformed of a single type of matrix material, it is possible, if notdesirable under some circumstances, to formulate contiguous layers ofthe microparticles from different matrix materials. Still further it ispossible that under some circumstances, it may be desirable to delivermore than one antibody or antigen to the internal reproductive organs totreat more than one condition. Thus, for instance, monolithicmicroparticles could be prepared and delivered containing two differentantibodies to passively treat two different diseases. In fact, it may bedesirable under some circumstances to actively immunize a patientagainst one disorder and simultaneously passively immunize the patientagainst a second disorder with antigen and antibody delivered in thesame microparticles. Of course, when more than one antigen and/orantibody is combined in one microparticle where they may be in contactand not in different layers of a microparticle, they must not react witheach other. A very similar situation exists where it may be desirable toinclude more than one type of estrogen or progestin in the samemicroparticle. The above embodiments only describe several of manypossible microparticle configurations.

With regard to the physical size or shape of the microparticles, themicroparticles can assume any possible shape ranging from ordered shapessuch as spherical or oval to irregular shapes. The shape of themicroparticles is not a factor in microparticle transport.

The size of the microparticles is important insofar as themicroparticles must possess sperm surrogate activity such that they canbe conveyed by the natural transport mechanism of the reproductiveorgans upward from the cervix into the uterus and eventually into thefallopian tubes. If the microparticles are too large, they will causecontractions of the cervix which will expel the microparticles.Microparticles which are too small will not be conveyed upward into theinternal reproductive organs. Usually, the microparticles range from 10to 100 μm, preferably 20 to 70 μm, most preferably 20-60 μm. While themechanism of microparticle transport is not known precisely, severalfactors are known which are important for transport. The cervical mucousis a type of aqueous hydrogel which is dynamic in that the viscosity andflow characteristics range during the monthly cycle of the reproductiveorgans. If transport of the microparticles is to be achieved, the matrixmaterial of the microparticles must be compatible with the cervicalmucous. The compatibility of the microparticles is a function of notonly the chemical nature of the matrix material of the microparticles,but also the size of the microparticles. The microparticles must bemiscible with the cervical mucous. Transport of the microparticlesacross the cervix, just as in the case of the sperm, is achieved onlywhen the cervix is prepared to transport the microcapsules. Accordingly,the microparticles containing antigen or antibody must be deposited inthe vagina when the cervix is ready to transport the microparticles.Once the cervix is ready for transport, synchronized muscle contractionsof the cervis propel the microparticles into the uterus where eventuallysome of the microparticles are conveyed through the uterus into thefallopian tubes, although the important objective of the invention isthat the microparticles should cross through the cervix into the uterus.The muscle contractions of the cervix are controlled by the naturalrelease of hormones at the appropriate time of the monthly female cyclewhich as discussed above is at midcycle. Alternatively, as discussed atlength above, the transport mechanism can be stimulated by theadministration of cycle regulatory hormone.

In the preparation of the antibody or antigen containing microparticlesessentially any known antigen or antibody can be incorporated in themicroparticles although those of particular use in the treatment ofconditions and diseases of the internal reproductive organs arepreferably used. Antibodies of the same type have the same biochemicalstructure regardless of what antigen they react with. Therefore, thesame process can be used to incorporate any type of antibody inmicroparticles regardless of their specificity. Suitable types ofantigens which can be incorporated in the present microparticles includebacterial and viral pathogens of man and animals, however, enzymes andother biological factors involved in the reproductive process can alsobe used. Suitable pathogenic antigens include Neisseria gonorrhea,Mycobacterium tuberculosis, Herpes virus (humonis, types 1 and 2),Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilusvaginalis, Group B Streptococcus ecoli, Microplasma hominis, Hemophilusducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum,Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis,Campylobacter fetus, Campylobacter fetus intestinalis, Leptospirapomona, Listeria monocytogenes, Brucella ovis, Equine herpes virus 1,Equine arteritis virus, IBR-IBP virus, BVD-MB virus, Chlamydia psittaci,Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillusequuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonasaeruginosa, Corynebacterium equi, Corynebacterium pyogenes,Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillusfumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi,Clostridium tetani.

Suitable examples of enzymes that may be involved in the reproductiveprocess include ribonuclease, neuramidinase, trypsin, glycogenphosphorylase, sperm lactic dehydrogenase, sperm hyaluronidase,adenossinetriphosphatase, alkaline phosphatase, alkaline phosphataseesterase, amino peptidase, trypsin chymotrypsin, amylase, muramidase,acrosomal proteinase, diesterase, glutamic acid dehydrogenase, succinicacid dehydrogenase, beta-glycophosphatase, lipase, ATP-ase alpha-peptategamma-glutamylotrans peptidase, sterol-3-beta-ol-dehydrogenase,DPN-di-aprorase.

Suitable examples of hormones acting as antigens include human chorionicgonadotrophin hormones, human placental lactogen, progesterone,estradiol and the like. Suitable antigens include those known asembryonic cellular antigens which occur on the cellular surface of thetrophoblast and are unique to the trophoblast. In addition to the abovementioned pathogens, mixture of pathogens which can infect the femalereproductive organs also can be incorporated in microparticles. Statedsimply any pathogen can be used to produce a vaccine which could then beused to immunize the cervix, uterus and fallopian tubes by the presenttechnique.

Examples of antibodies for passive immunization which can beincorporated in microparticles include those which correspond to all ofthe above described antigens which are effective for activeimmunization.

When the microparticles of the present invention are administered to asubject, they are administered in an amount sufficient to elicit aneffective level of response over a period of time desired. For treatmentof the cervix, uterus and/or fallopian tubes a suitable dose range foran antigen for the role of primary active immunization would be 0.5 to 1mg of antigen per day over a 7-14 day period. The dosage range requiredfor a booster immunization would vary from 0.5 to 1 mg per day over a 24hour time span. With regard to passive immunization via antibodyadministration, the weight of antibody administered does not necessarilydirectly relate to the therapeutic effect realized. The important factorin terms of dosage for passive immunization is the titer of the antibodyor the biological potency. The titer of an antibody refers to themaximum dilution of the antibody which elicits an effect in a testsituation. Two different preparations of antibody are not equallycomparable on a weight basis because they have different biologicalpotencies. An immunological titer of 1:500 is the minimum biologicalpotency for any antibody to be administered by the process of thepresent invention. Moreover, the rate at which the immumoglobulin orantibody should be delivered to the cervix, uterus and fallopian tubesshould not exceed 0.1 mg of antibody per day. Any dose rate less thanthis level which is effective in eliciting a therapeutic response isacceptable. Dosage rates greater than this level are unacceptablebecause the larger dose may cause a sensitization reaction against theantibody. That is, if the dosage rate is too great, the passivelyadministered antibody might function as an antigen thereby stimulatingthe production of antibodies in the host that would react with theadministered antibodies.

As described above, the matrix material in which the antigen or antibodyis incorporated is an important consideration. Three factors predominatein the selection of a matrix material which are the biocompatibility ofthe matrix material with the mucosal fluids, the permeability of thematrix material and the ability of the matrix material to degradebiologically by a mechanism such as hydrolysis so that no matrixmaterial residues remain after transport and deterioration of themicroparticles in the uterus and fallopian tubes. The release of antigenor antibody from the microparticles may occur by diffusion of the agentthrough the entrapping matrix material or by erosion of the matrixmaterial or by a combination of both factors. Suitable polymers asmatrix materials include polyglycolic acid, polylactic acid, as well ascopolymers of glycolic and lactic acid, and glycerol mono- anddistearate. The preferred matrix materials, however, are polylactic acidand polyglycolic acid. The aliphatic polyesters described above degradebiologically by hydrolysis under physiological conditions and areconverted to monomeric glycolic and lactic acids. The rate ofdegradation of the polymer in the body preferably occurs as soon aspossible after the drug is released and is related to the rate ofhydrolysis of the ester linkages which is, in turn, related to thesurface area of the microcapsule or device, the crystallinity of thepolymer, and the inherent hydraulic stability of the polyester.

The molecular weight of the particular polymer chosen is not a criticalfactor in the manufacture and use of the microcapsules. While anincrease in the molecular weight of the polymer may gradually retard therate of release of therapeutic agent(s) from the microcapsules and thusaffect the dosage level administered, these factors may easily becompensated for by determining the rate of release of therapeuticagent(s) from the microparticles by in vivo or in vitro measurements andthen adjusting the amount of microparticles administered in view of theresults obtained from the release measurements.

The antigen or antibody containing microcapsules can be convenientlyprepared by any well known procedure used in the past for thepreparation of microparticles containing a pharmaceutical material.While the amount of antigen or antibody, and cycle regulatory hormone,if it is to be present, is not critical, normally, the microparticlescontain from about 10 wt.% to 60 wt.%, preferably 10 wt.% to 50 wt.%,most preferably 10 wt.% to 25 wt.% of antibody or antigen. The methodsselected for preparing the microparticles are not critical although theywill vary principally depending upon the type of microparticles to beprepared, i.e. whether the microparticles are to be monolithic, ormanufactured such that a core of pharmaceutical material is surroundedby a wall of encapsulated matrix material or manufactured in a mannersuch that an onion-skin type of structure results with alternatinglayers of matrix material and pharmaceutical material alone or in amatrix material.

In the manufacture of the microparticles containing antigen or antibodyand/or a menstrual cycle regulatory hormone, any conventional method offorming the microparticles can be used. The selection of a particularmethod chiefly depends upon the technical requirements of the matrixmaterial and the particular manner in which the microparticles areintended to be used.

Generally, microencapsulation processes can be classified according tothe three principal types of: (1) phase-separation methods includingaqueous and organic phase separation processes, melt dispersion andspray drying; (2) interfacial reactions including interfacialpolymerization, in situ polymerization and chemical vapor deposition;and (3) physical methods, including fluidized-bed spray coating, multi-and single-orifice centrifugal coating, electrostatic coating andphysical vapor deposition.

Phase separation method, as the term implies, rely on differentialsolubility characteristics that cause a wall- or shell-forming matrixmaterial to separate from solution or suspension and deposit aroundparticles or droplets of the therapeutic agent(s) to be encapsulated.The separation itself may be brought about physically, as by theaddition of a non-solvent or by a change in temperature, or chemically,as by a change in pH.

An organic phase-separation process usually employs a dispersion or anemulsion of the therapeutic agent(s) in a solution or ahigh-molecular-weight polymer in an organic solvent. To this mixture isadded a non-solvent or liquid polymer that causes thehigh-molecular-weight polymer to separate from solution and collect as ashell around the suspended therapeutic agent(s). The shell, stillswollen with solvent, is then hardened by a further addition ofnon-solvent or by some other process that strengthens the shell andimproves the barrier properties.

Typically, an aqueous solution or suspension of a lipophobic antigen orantibody and/or menstrual cycle regulatory hormone is added to anon-aqueous solution of a suitable matrix polymer, and the mixture isagitated to cause the formation of a water-in-oil emulsion. Dependingupon its solubility in water, the agent may be present at aconcentration of 5 to 50% in the aqueous phase, which may be 5 to 20% byweight of the total mixture. The external organic phase may contain 5 to10% of the matrix polymer. Usually, however, the ratio of agent in theinternal phase (aqueous solution or suspension) to polymer is 2:1 to1:4. The polymer must be a good film-former; that is, it most possessadequate strength and toughness.

An aqueous phase separation process employs a dispersion or an emulsionof a water-insoluble therapeutic agent(s) in an aqueous solution ofdispersion of a polymer. The polymer is caused to separate as gelparticles; these collect around the therapeutic agent to form a shell;the shell is hardened; and the microparticles are isolated. In theconservation process, which is the most common of the aqueousphase-separation processes, the water-insoluble therapeutic agent, whichmay be in the form of particles or droplets, is usually dispersed in anaqueous sol of a hydrophilic colloid which becomes ionized in water; asecond sol of opposite charge is added; and the mixture is caused to gelby a dilution with water, an addition of salt, an adjustment of pH, or achange in temperature, or by combination of these. Appropriateconditions of conservation are usually determined experimentally,because the various polymers, possible for use, differ significantly inphysical and chemical properties according to source and method ofisolation or preparation. A region of coacervation is determined bycombining solutions or sols of two polymers at various concentrations,temperatures and levels of pH, and observing the conditions required forgelation. From these determinations can be drawn a ternary phasediagram, showing the area of compatibility and the region ofcoacervation, at a given temperature and pH. The changes inconcentration, temperature or pH to effect gelation will then becomeapparent.

Each preparation of microparticles requires carful control ofconditions, and somewhat different conditions are required for varioustherapeutic agents. The degree of agitation, for example, affects thesize of emulsion droplets, and the surface properties of the dropletsmay require alterations in the procedures to insure deposition of matrixmaterial about the droplets and to minimize formation of particles notparticipating in microencapsulation. The volume of water added in thedilution step is not critical, but generally larger volumes are requiredto maintain a stable emulsion when larger droplets are encapsulated.

The above phase separation can be adapted to an alternate technique inwhich the first step of forming a stable emulsion or suspension of themedicinal or therapeutic agent is accomplished by dispersing the agentin a solution of the matrix material. Thereafter, the emulsion is addeddropwise to a non-solvent with stirring to precipitate the polymercoating material to form microparticles.

Another type of phase separation technique is the melt-dispersionmicroencapsulation technique. This method can be used with a widevariety of medicinal or therapeutic agents. Usually a heat-liquifiable,waxy coating material, preferably of a low-melting wax such as glyceroldistearate is suspended in an inert liquid such as a silicone oil or afluorocarbon in which neither the wax nor the material to beencapsulated is appreciably soluble. The mixture is heated and stirredvigorously to melt and emulsify the wax. The therapeutic agent which hasbeen powdered and screened to the desired size range, and the waxycoating material are dispersed with high shear agitation, and theliquefied wax coats the therapeutic agent to form the waxy liquid-coatedmicroparticles. Thereafter, the formed microparticles ae solidified bycontinued agitation which cools the particles. The microparticles arethen isolated by filtration and dried as described earlier.

The second major method of forming the microparticles is by interfacialmicroencapsulation which involves bringing two reactants together at areaction interface where polycondensation of the reactants, usuallymonomers, occurs to form a thin, insoluble polymeric film. One techniqueof establising the interface for the encapsulation process is thedispersion or emulsification of the therapeutic agent with one of thereactants which form the condensation polymer in a continuous phasecontaining the second reactants.

The third major category of encapsulation techniques which is especiallyapplicable to a variety of medicinal or therapeutic agents and coatingmaterials is physical microencapsulation. The physicalmicroencapsulation techniques are characterized by the continuousenvelopment of particles or droplets of a medicinal or therapeutic agentin a fluid film, as a melt or solution of the coating material, in anapparatus containing coaxially or sequentially-spaced orifices.Thereafter, the fluid coating is hardened by a standard coolingtechnique or by solvent evaporation.

Among the physical methods for microencapulsation are those that involvethe passage of liquid or solid core material through a liquid matrixmaterial. The stream is disrupted by some means to cause the formationof liquid-coated droplets or particles, and the resulting particles arecooled or otherwise treated to solidify the shell material. For example,an aqueous solution of a therapeutic agent is aspirated into a rapidlyflowing stream of molten glycerol disterate, and the mixture is ejectedthrough a fine nozzle. On emergence from the nozzle, the liquid streamdisintegrates into droplets, each consisting of an aqueous coresurrounded by liquid wax. As these fall through air, the shells cool andsolidify, and microparticles result. In another version of this process,the impelling force is supplied by a rotating member, which ejects thecore material centrifugally through the shell-forming liquid.

The variations of these and other processes of microencapsulation aremany. As is readily apparent to those skilled in the art, no one processnor any single set of conditions is applicable to all therapeuticagents, but instead a useful process is chosen and the conditionsoptimized to achieve the desired results with a specific agent.

Microcapsules containing medicinal or therapeutic agents can bedelivered in the vagina by a variety of methods. The preferred method isto incorporate a fixed number of microcapsules into a container designedfor easy hand insertion into the vagina. The insertion container shouldbe made of a biodegradable material that dissolves within minutes afterplacement in the vagina, thus, releasing the microcapsules.Pharmaceutical type gelatin capsules can be conveniently used as adelivery system for the microcapsules. The dose level can be varied byincreasing or decreasing the number of microcapsules in the deliverydevice. Of course, any number of other methods of variations, or thispreferred method might be used. For example the microcapsules could bemolded into a solid vaginal suppository by using an appropriatesuspension medium such as gelatin. Creams, jellies, foams, or liquidsmight be used as a suspension medium for microcapsules. Preparations ofthis type could be placed in the vagina using a loadable syringe or sometype of pressurized vaginal inserter. A variety of different types ofapplicators for administering pharmaceutical agents to the vagina andrectum are in common use. These consist of two parts; a nozzle designfor easy insertion into the vagina, and a hand held implement used toproject the preparation into the vagina. Syringes, squeeze bulbs,squeeze tubes and aerosol containers are examples of implements that canbe used to generate the force necessary to propel the preparation intothe vagina.

Vaginal suppositories offer the simplest, most direct method ofapplication. The microparticles are inserted into the lower half of apreformed gelatin shell. The margin of the shell is then moistened withwater and the upper half of the shell is joined to the lower half tocomplete formation of the suppository Four, eight and twelve graingelatin capsules can be used in this manner depending upon the dosage ofmicrocapsules desired. Suppositories of other materials such as jellies,creams, foams or aerosols can also be used as the delivery system formicrocapsules. The dose can be strictly regulated by including a fixednumber of microcapsules in the suppository preparation, and the carrierdevice can be applied by hand. Another advantage is that by using ahollow container such as a gelatin capsule, special suspension mediawhich might adversely affect the migration of the microcapsules are notneeded.

The primary limitation for the generation of passive immunization in asubject by the administration of antibodies in clincal medicine is thatantibodies produced in animals quite often cause serum sickness oranaphylaxis when injected into human recipients. However, the localdelivery technique of the present invention in which microencapsulatedantibodies are transported into the internal female reproductive organscircumvents this problem because not only are smaller dosages ofantibodies required, but also systemic administration of antibodies isavoided. It is acknowledged that the use of humans as antibody donorscan circumvent this problem. However, human donors cannot be used safelywhen immunization affects their own physiology or necessitate the use ofantigens which are toxic. A distinguishing feature between active andpassive immunization is that the natural elimination of passivelyadministered antibodies from a subject renders this approach temporaryand reversible, whereas active immunization of a subject is usuallypermanent and non-reversible.

Any type of reaction between administered antibody and antigen withinthe local environment of the internal reproductive organs to elicit apassive immunization response is within the scope of the presentinvention. For example, antibodies effective against any type ofbacterial or viral pathogen can be used in the local treatment ofinfections in the vagina, cervix, uterus and fallopian tubes. Similarly,antibodies produced against sperm, egg, products of conception andbiological factors in the reproductive fluids including hormones such asHCG and enzymes can be employed to prevent pregnancy. One potentiallyvery important application of the present invention is a method oftreating the veneral disease, gonorrhea, which is caused by themicroorganism, Neisseria gonorrhea. This microorganism lives andproliferates in cavities in the cervix, uterus and fallopian tubes ofinfected women. Thus, the present invention provides a technique ofgenerating a passive immunization in an infected host against thisdisease. It is noteworthy to emphasize at this point that there is noknown method of immunization against gonorrhea because standard methodsof immunization are not effective since these techniques induce systemicantibodies which are not secreted into the various parts of the internalfemale reproductive organs. Other diseases which can be treated by thepassive immunization technique of the present invention includesyphillis, simplex herpes viral infections, yeast infections,trichomoniasis bacterial infections and the like.

Still another aspect of passive immunization within the scope of thepresent invention is the use of antibodies to reduce female fertility.The human preimplantation embryo produces and secretes a hormone calledchorionic gonadotrophin hormone (HCG), which is necessary forimplantation of the embryo into the uterus. It is known that antibodiesagainst HCG neutralize the functions of this hormone and preventimplantation of the embryo from occuring. Accordingly, antibodieseffective against HCG can be introduced into the internal femalereproductive organs by the the technique of the present invention toprevent pregnancy. This technique can be extrapolated to the use ofantibodies against sperm, egg, products of conception and a wide varietyof enzymes and hormones which can be found in the fluids of thereproductive tract.

In some instances active immunization is more advantageous than passiveimmunization with an obvious example being active immunization forpermanent protection against infectious diseases. Thus, when an antigenis incorporated within microparticles which can be delivered to thecervix, uterus and fallopian tubes, the delivered antigen induces theformation and secretion of specific antibodies by these organs. Thesecreted antibodies not only provide the desired immunological effect,but also are structurally and fundamentally unique from the type ofantibody produced in response to systemic immunization. Systemicantibodies are not secreted by the reproductive organs, and it is forthis reason that systemic immunization is not an effective way ofgenerating antibodies in the fluids of the cervix, uterus and fallopiantubes. Thus, standard methods of immunization are not effective in theprevention of infections of the internal female organs or forcontrolling fertility. In the present invention, on the other hand, notonly are antigens delivered directly to the internal female organs, butthe mode of release of antigen from the microparticles varying from asudden release to a sustained release ensures that the antigen can beadministered as desired thus ensuring sensitization.

In a typical example of active immunization, antigens from themicroorganism, Neisseria gonorrhea, for instance, are incorporated in apolymer such as polylactic acid. The microparticles are thenadministered into the vagina of a subject where they are then conveyedacross the cervix into the uterus. The microparticles release antigen atthe desired rate perhaps in the cervix as the microparticles areconveyed into the uterus. The antigen sensitizes the secretory tissuesof the internal organs which respond by producing protective antibodies.The secreted antibodies form a protective fluid coating along thesurfaces of the internal organs which protects the subject against aninvasion and infection of N. gonorrhea microorganisms. The sametechnique can be employed to immunize a subject against other bacterialand viral infections of the internal female reproductive organs.

Another aspect of active immunization pertains to fertility. In thiscase, sperm antigens are delivered by transport of antigen containingmicrocapsules into the cervix, uterus and fallopian tubes. The antigenwhich is slowly released over a sustained period of time, stimulates thesecretory tissues of the organs to secrete protective antibodies in thefluid layer which coats the internal organs which essentially are thecervix, uterus and fallopian tubes. After copulation and deposition ofsperm in the vagina, antibodies in the cervical mucous causeagglutination of the sperm in the cervix and prevent further transportof the sperm into the uterus. Antibodies against sperm also inactivatesperm by techniques other than agglutination.

There are many recognized advantages to fertility control byimmunization. The most obvious benefit is that immunization with ananti-fertility vaccine could provide sustained fertility control. Animportant advantage of the local administration technique of the presentinvention is that the vaccine could be self-administered at low cost.Recently, several antigens have been isolated and identified which areunique to the reproductive process which will induce an anti-fertilityimmune response. These antigens include those of the blastocyst, theovum, the sperm, non-hormonal placental antigens and trophoblastichormonal antigens.

The present concept of basing fertility control upon the local anddirect administration of antigens to the internal female reproductiveorgans is founded on the assumption that a high concentration ofantibodies within the reproductive tract may be more efficacious andsafe for inhibition of sperm or blastocyst vitality than systemicimmunization. It is believed that anti-sperm or anti-trophoblastichormone immunity interferes with the reproductive process in the femalegenital tract before or during embryonic implantation. In fact, it isknown that when HCG antigens are administered systemically, systemicantibodies may cross-react with other tissues of the body thereby givingrise to detrimental side-effects. The risk of the systemic side-effectsprecludes the use of this method for controlling fertility in humans.However, if indeed local immunity of the reproductive organs can beincreased in the absence of a systemic immune response, then many of thecomplications associated with systemic immunization can be avoided.Furthermore, the present invention has the advantage that cyclicoverdosing and underdosing which are inherent in conventional methods ofadministering drugs can be obviated by the sustained release of antigenor antibody from microparticles. Thus, the present technique affords ameans for effecting a pharmacological response with a minimum dose ofdrugs.

Having now generally described the invention, a more completeunderstanding can be obtained by reference to certain specific exampleswhich are included for purposes of illustration only and are notintended to be limiting unless otherwise specified.

EXAMPLE 1 Preparation of Progesterone Containing Polylactic AcidMicrocapsules

A 2.5 g amount of progesterone and 10.0 g of d,l-polylactic acid weredissolved in 38 g of methylene chloride. The resulting viscous solutionwas poured into a 250 ml kettle containing 120 ml of a 5 wt.% aqueouspolyvinylalcohol solution. The dispersion obtained was stirred at about2000 rpm until a stable emulsion had formed with the droplets being inthe range of 50 to 100 μm in diameter. A vacuum was applied to theemulsion until it began to foam and then the rate of stirring wasreduced to 600 rpm. After two hours, most of the methylene chloride hadevaporated. Moreover, continuous stirring was not required to preventthe embryonic microcapsules from agglomerating. Thereafter, the emulsionwas centrifuged, the aqueous polyvinylalcohol solution was decanted andthe microcapsules were resuspended in 150 ml of deionized water. Forabout 18 hours thereafter a vacuum was continually applied to thestirred aqueous suspension. Thereafter, the suspension was centrifugedand the microcapsules obtained were washed with water and then collectedby vacuum filtration. The microcapsules were dried at room temperatureunder a hard vacuum overnight, and then they were sieved whereby afraction ranging between 43 and 61 μm was obtained. By this proceduremicrocapsules containing 22±1.5 wt.% progesterone were obtained.

EXAMPLE 2

The procedure of Example 1 was followed to the extent that theingredients were mixed and stirred in the aqueous polyvinylalcohol. Avacuum was applied to the stirred dispersion and after about 2 hours,when 90 wt.% of the solvent had been removed, the procedure wasinterrupted. The suspension was centrifuged and the microcapsules wereobtained after decantation. The microcapsules were resuspended indeionized water which did not contain a dispersing agent. A vacuum wasreapplied to the suspended microcapsules and the procedure was continuedto completion. By this technique the encapsulation efficiency was 100%.

EXAMPLE 3 Preparation of Progesterone containing Glycerol MonostearateMicrocapsules

A 1.0 g amount of progesterone was added to 4 g of molten glycerolmonostearate and a portion of the molten mixture was poured into thereservoir of a melt sprayer and heated to 167° C. The flow of nitrogeninto the device to effect cooling was 60 liters per minute, while theflow of nitrogen into the sprayer to aerosolize the molten mixture wasadjusted to the maximum rate of 5.75 liters per minute. The aerosol wassprayed intermittently, and microcapsules were collected and sieved,whereby a size fraction ranging between 43 and 61 μm was collected. Themicrocapsules produced by this procedure were spherical and contained a20 wt.% theoretical loading of progesterone.

EXAMPLE 4 A. Preparation of Antigen Containing Microcapsules

An amount of 1.5 g to 2.5 g of a poly (glycolide-lactide) excipient wasdissolved in approximately 100 g of methylene chloride. The copolymercomprised a 50:50 mole ratio of glycolide to lactide. The copolymersolution was placed in a 200-ml Bantamware resin kettle equipped with atrue-bore stirrer and a 1.5 inch teflon turbine impeller driven by aFisher "StediSpeed" motor. To this solution was added 0.5 g of anaqueous dispersion of pneumococcus type-III antigen, 0.05 g of anaqueous solution of Herpes simplex antigen or a solution prepared bydissolving 0.15 to 0.30 g of Anti-β-hCG cow antibody in approximately0.75 g of deionized water. During the addition of the antigen orantibody material, the contents of the resin kettle were stirred at2100-3000 rpm to form a water-in-oil emulsion. The water micro dropletswhich formed contained dissolved biological agent and had particle sizediameters ranging from 5-60 μm. Stirring of the emulsion was continuedand 40-50 ml of silicone oil (Dow Corning 200, 350 cs) was pumped intothe resin kettle at a rate of 1-2 ml/min. The silicone oil promotedcopolymer phase separation because of copolymer-polymer incompatibility.The copolymer was deposited as droplets of solvent-swollen copolymeronto the surface of the water microdroplets. As more silicone oil wasadded, more copolymer droplets accumulated until they coalesced to forma continuous film around the water micro droplets. The copolymer filmwas then hardened by pouring the contents of the resin kettle into abeaker containing about 2000 ml of heptane stirred at 600-1000 rpm witha 2.5 inch stainless steel impeller. This process hardened themicrocapsules since heptane is a non-solvent for the copolymer and asolvent for methylene chloride and silicone oil. The heptane extractedmethylene chloride from the solvent-swollen walls of the microcapsules,and washed the silicone oil from the surface of the microcapsules. Afterbeing stirred in heptane for about 30 minutes, the microcapsules werecollected and washed several times with more heptane to remove residualquantities of methylene chloride and silicone oil. The hardenedmicrocapsules were then isolated by vacuum filtration through a Buchnerfunnel and Whatman number 41 filter paper, and were dried for about 24hours in a vacuum chamber maintained at room temperature. By thistechnique free flowing particles having diameters ranging from 10 to 90μm were obtained. In this manner three batches of microcapsules wereprepared within the size range of 10-90 μm of which the first batch(microcapsule system 1) contained the antigen of pneumococcus, type III,the second batch (microcapsule system 2) contained the antigen fromHerpes simplex virus, and the third batch (microcapsule 3) contained theprotein antigen or antibody.

B. Treatment Of Test Animals

The following animal experiments were conducted to demonstrate theutility of microcapsule systems 1, 2 and 3 in immunizing the internalfemale reproductive organs. For these experiments adult female rabbitswere used; the standard animal model for immunological studies. Fiverabbits were assigned to each of three experimental groups and prior totreatment, blood samples and washings from the vagina of each rabbitwere obtained. The blood serum and vaginal washes from each animal weretested for the presence of antibodies against pneumococcus, type IIIantigen, Herpes simplex virus antigen, and Bovine IgG. The serum andvaginal washings from each rabbit were also tested for immune reactivityagainst the β sub unit of hCG. Only rabbits which exhibited negativeresults in each of the tests were selected for inclusion in the tests ofthe present invention. Standard immunological tests were used fordemonstrating the existance of antibodies against the bacteria, virusand protein, and for the demonstration of antibodies which react withthe β sub unit of hCG.

Each of the adult female rabbits was vaginally administered a 5 mgquantity of a batch of microcapsules obtained from microcapsule system1, 2 or 3. Five rabbits were treated with each of the three differentsystems. Two weeks following treatment, vaginal washes were obtainedfrom the rabbits and tested for the presence of antibodies againstpneumococcus, type III bacteria (treatment group 1), Herpes simplexvirus (treatment group 2), and Bovine IgG (treatment group 3).

The results of the experiments are shown in Table 1 below. The tableshows that the pretreatment vaginal wash samples from all fifteenrabbits tested contained no demonstrable antibody against pneumococcus,type III bacteria, Herpes simplex type virus or Bovine IgG. The tablefurther shows that following treatment by intravaginal insertion of 5 mgof microcapsules containing pneumococcus, type 3 bacteria, thepost-treatment vaginal washes from rabbits 1-5 exhibited positive titersagainst pneumococcus, type III bacteria with negative titers againstHerpes simplex virus and Bovine IgG. he vaginal washes obtained fromrabbits 1-5 also lack demonstrable immunological reactivity against theβ sub unit of hCG. The results clearly show that rabbits 1-5 respondedto the intravaginal challenge with microencapsulated pneumococcus, typeIII bacteria by providing antibodies against the bacterial antigens.

With respect to rabbits 6-10, the vaginal washes from these animalsexhibited negative titers to all three antigens and no demonstrableimmunological activity against hCG. The post-treatment vaginal washesfrom rabbits 6-10 exhibited positive antibody titers against Herpessimplex virus and negative titers against pneumococcus, type III andBovine IgG. None of the samples from the rabbits contained anydemonstrable immunological activity against the β sub unit of HCG. Theresults clearly indicate that intravaginal installation of microcapsulescontaining Herpes simplex virus antigen caused immunization againstHerpes simplex virus.

The pretreatment vaginal washes from rabbits 11-15 contained negativeantibody titers against pneumococcus, type III, Herpes simplex virus,and bovine IgG and lacked demonstrable immunological reactivity againstthe beta subunit of hCG. The post-treatment samples had positive titersagainst bovine IgG, and negative titers against pneumococcus, type IIIbacteria and herpes simplex virus. It is significant to note that thepost-treatment vaginal washes in rabbits 11-15 also containeddemonstrable antibody titers against the beta subunit of hCG. This lastexample illustrates two different applications of the vaginalmicrocapsule system. On the one hand it demonstrates that the systemprovides an effective method for the delivery of a protein antigen; inthis case, bovine IgG, the results being positive titer against thebovine IgG. The protein in this example is an antibody which has its ownimmunological reactivity. The demonstration of this antibody in thepost-treatment vaginal wash illustrates the use of the microcapsulesystem for the delivery of antibodies; thus, establishing the utility ofthe system for passive immunization.

                                      TABLE I                                     __________________________________________________________________________    SUMMARY OF RESULTS                                                                      Vaginal Wash Titers                                                           Bacteria   Virus      Protein    Antibody                                Treat.                                                                             Pneumococcus                                                                             Herpes     Bovine     Anti-                              Rabbit                                                                             System                                                                             Type 3     Simplex    IgG        hCG                                Number                                                                             Number                                                                             Pre-treat.                                                                         Post-treat.                                                                         Pre-treat.                                                                         Post-treat.                                                                         Pre-treat.                                                                         Post-treat.                                                                         Pre-treat.                                                                         Post-treat.                   __________________________________________________________________________    1    1    -    +     -    -     -    -     -    -                             2    1    -    +     -    -     -    -     -    -                             3    1    -    +     -    -     -    -     -    -                             4    1    -    +     -    -     -    -     -    -                             5    1    -    +     -    -     -    -     -    -                             6    2    -    -     -    +     -    -     -    -                             7    2    -    -     -    +     -    -     -    -                             8    2    -    -     -    +     -    -     -    -                             9    2    -    -     -    +     -    -     -    -                             10   2    -    -     -    -     -    -     -    -                             11   3    -    -     -    -     -    +     -    +                             12   3    -    -     -    -     -    +     -    +                             13   3    -    -     -    -     -    +     -    +                             14   3    -    -     -    -     -    +     -    +                             15   3    -    -     -    -     -    +     -    +                             __________________________________________________________________________

Having now fully described this invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or the scope of theinvention as set forth herein.

What is claimed as new and intended to be secured by Letters Patentis:
 1. Anti-B-HCG containing microparticles for the immunization of theinternal female reproductive organs, said microparticles containing abiologically effective amount of anti-B-HCG incorporated in a matrixmaterial which is biocompatible and biologically degradable, saidmicrocapsules being capable of being transported after deposition in thevagina by the natural transport mechanism of the internal femalereproductive organs across the cervix into the uterus whereinimmunization is effected within said internal female organs, saidmicroparticles having a diameter of from 10 to 100 μm.
 2. The anti-B-HCGcontaining microparticles of claim 1, wherein the said microparticleshave a diameter of from 20 to 70 μm.
 3. The anti-B-HCG containingmicroparticles of claim 1, wherein the said microparticles have adiameter of from 20 to 60 μm.
 4. The anti-B-HCG containingmicroparticles of claim 1, wherein the said microparticles comprise acentral anti-B-HCG containing core surrounded by an outer shell ofmatrix material free of anti-B-HCG.
 5. The anti-B-HCG containingmicroparticles of claim 4, wherein said outer shell of themicroparticles is resistant to the acidic pH environment of the vaginaand susceptible to breakdown when exposed to the neutral pH of thecervix.
 6. The anti-B-HCG containing microparticles of claim 4, saidmicroparticles having a configuration comprising alternate layers ofanti-B-HCG containing layers and anti-B-HCG free matrix material.
 7. Theanti-B-HCG containing microparticles of claim 1, said microparticlescontaining from 10 weight % to 60 weight % of anti-B-HCG.
 8. Theanti-B-HCG containing microparticles of claim 1, said microparticlescontaining from 10 weight % to 50 weight % of anti-B-HCG.
 9. Theanti-B-HCG containing microparticles of claim 1, said microparticlescontaining from 10 weight % to 25 weight % of anti-B-HCG.
 10. Theanti-B-HCG containing microparticles of claim 4, said microparticlescontaining from 10 weight % to 60 weight % of anti-B-HCG.
 11. Theanti-B-HCG containing microparticles of claim 4, said microparticlescontaining from 10 weight % to 50 weight % of anti-B-HCG.
 12. Theanti-B-HCG containing microparticles of claim 4, said microparticlescontaining from 10 weight % to 25 weight % of anti-B-HCG.
 13. Anti-B-HCGcontaining microparticles for the immunization of the internal femalereproductive organs, which comprise microparticles having a size of from10 to 100 μm and containing a biologically effective amount ofanti-B-HCG incorporated in a matrix material which is biocompatible andbiologically degradable, said microparticles being capable of beingtransported after deposition in the vagina by the natural transportmechanism of the internal female reproductive organs across the cervixinto the uterus wherein immunization is effected within said internalfemale reproductive organs.
 14. The microparticles of claim 13, whereinsaid microparticles further contain a menstrual cycle regulatory hormonewhich stimulates said natural transport mechanism.
 15. The microcapsulesof claim 14, wherein said hormone is estradiol or progesterone.
 16. Themicroparticles of claim 13, wherein said microparticles are of a sizeranging from 20 to 70 μm.
 17. The microparticles of claim 13, whereinsaid matrix material is polylactic acid, polyglycolic acid, orcopolymers of glycolic and lactic acids.
 18. The microparticles of claim13, wherein said microparticles are of a monolithic structure in whichsaid antibody is dispersed throughout the matrix material.
 19. Themicroparticles of claim 13, wherein said microparticles contain from 10wt.% to 60 wt.% of said antibody.